Abstract

Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. We have applied this strategy to develop an autoluminescent Ca2+ indicator, BRAC, which is composed of Ca2+-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. With this BRAC, we succeeded visualization of Ca2+ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca2+-independent signal drifts due to change in cell shape, focus shift, etc. Taking advantage of the bioluminescence imaging property that does not require external excitation light, BRAC might become a powerful tool applicable in conjunction with so-called optogenetic technology by which we can control cellular and protein function by light illumination.

Highlights

  • Indicator was developed based on split RLuc and Gluc [8,9]. Signal change of these indicators is based on complementation of the N- and C-terminal halves of the split luciferase via Ca2+-. Bioluminescent proteins such as luciferase are a powerful tool for monitoring biological processes including gene expression in living organisms since bioluminescent signals can be acquired without an external light source; bioluminescence imaging is completely free from phototoxicity, photo-induced physiological reaction, and autofluorescence from the specimen, enabling signal detection from deep inside the tissue with high signal-to-noise ratio

  • Bioluminescence signals are too dim to be measured in real time, i.e., bioluminescence imaging generally requires longer exposure than fluorescence imaging that takes less than 1 second

  • EBAF-Y is based on the highly efficient bioluminescence resonance energy transfer (BRET) between enhanced Renilla reniformis luciferase (RLuc8) [2] and enhanced yellow fluorescent protein (EYFP). eBAF-Y emits a 3.5-fold brighter signal than RLuc8 so Venus is not located close to RLuc8 and only weak emission can be seen from Venus due to low BRET efficiency

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Summary

Introduction

Bioluminescent proteins such as luciferase are a powerful tool for monitoring biological processes including gene expression in living organisms since bioluminescent signals can be acquired without an external light source; bioluminescence imaging is completely free from phototoxicity, photo-induced physiological reaction, and autofluorescence from the specimen, enabling signal detection from deep inside the tissue with high signal-to-noise ratio. Doi:10.1371/journal.pone.0009935.g001 induced interaction between CaM and M13 These indicators can detect Ca2+-dependent signal changes in living cells, they could not calibrate [Ca2+] because imaging of these indicators is based on single-emission measurement and the signal intensity change is highly dependent on the stoichiometric composition of the N- and C-terminal halves of the split luciferase in the cells. Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination

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